In humans, CPs are responsible for apoptosis, MHC Class II immune responses, prohormone processing and intracellular and extracellular matrix remodeling. As examples of CPs, there are: Actinidain, Bromelain, Calpains, Caspases, Cathepsins, Mir1-CPs, and Papain. CPs have also been recognized as critical enzymes in degenerative and autoimmune states (Zbigniew et al. 2007, Industrial Enzymes, book, 181-195). As an example, tumor cell invasion and metastasis are associated with the proteolytic activities of various types of proteases. Elevated expression of certain Cathepsins and diminished levels of their inhibitors have been reported to be involved in several human cancers, including brain, breast, gastric, glioma, prostate, lung, head and neck cancer and melanoma (Berdowska, Clinica Chimica Acta 342 (2004) 41-69). Beside cancers, Cathepsins have been reported to be involved in inflammatory diseases, such as inflammatory myopathies, rheumatoid arthritis and periodontitis.
Cysteine proteases of the papain family have been reported to play an important role in microbial (viral, bacterial) and parasitic infectious (Tong et al, 2002, Chem. Rev. 102, 4609-4626 and Han et al, 2005, Biochemistry 44, 10349-10359).
Caspases comprise a family of cysteine protease enzymes with a well-known role as key mediators in apoptosis signaling pathways and cell disassembly. Interleukin converting enzyme (ICE), also known as Caspase-1, was the first identified caspase and has a proinflammatory role. There is growing evidence demonstrating the role of caspases in very diverse pathologies. For instance it is known that proapoptotic caspases are involved in the pathogenesis of many cardiovascular disorders. Some proapoptotic caspases such as caspase-8 also possess non-apoptotic function that may contribute to tumor progression. Caspase-1 plays an important role in the response to pathogenic infection as well as in inflammatory and autoimmune disorders. In addition, caspase-1 activity is increased in retinas of diabetic patients and it constitutes a critical regulator of cardiomyocyte programmed cell death in the mammalian heart. Caspases also play a role in neurodegenerative diseases and trauma. For instance, it has been shown that the caspase-3 cascade is highly activated in traumatic spinal cord injury. Finally, the activation of caspase-1 and caspase-3 in Amyotrophic Lateral Sclerosis (ALS) patients and the activation of caspase-7, -8, and -9 in a mouse model at end stage of ALS have been reported. Increased levels of apoptosis and caspase activity (especially caspase-3) are reported to be frequently observed at sites of cellular damage in both acute (e.g. sepsis, myocardial infarction (MI), ischemic Stroke, spinal cord injury (SCI), traumatic brain injury (TBI)) and neurodegenerative disease (e.g. Alzheimer's, Parkinson's and Huntington's diseases, and multiple sclerosis (MS)). Caspase-6 is implicated in many neurodegenerative diseases including Alzheimer and Huntington's diseases. In Alzheimer's caspase-6 has been shown to cut amyloid precursor protein (APP) and Tau leading to a toxic fragment. In Huntington's disease, caspase-6 may be responsible for the type of Huntington fragments that lead to nerve cell death and symptoms.
Since caspases are involved in a number of diseases, several compounds and methods have been developed to inactivate them. For example, the broad irreversible caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) was protective and efficiently blocked death receptor-mediated liver injury in animal models (Rodriguez et al. (1996), J Exp Med. 1996 Nov. 1; 184(5):2067-72). Myocardial infarction and the resulting death of myocytes was ameliorated by z-VAD-fmk and related peptide inhibitors in animal models (Yaoita et al., 91998) Circulation 97: 276-281). There has been a lot of effort into identifying inhibitors of peptidase. For instance, Hanzlik and Thompson (J. Med. Chem. (1984), 27(6), 711-712) describe vinylogous amino acid esters for inactivating thiol proteases. Thompson et al. (J. Med. Chem. (1986), 29(1), 104-111) describe carboxyl-modified amino acids and peptides as protease inhibitors. Liu and Hanzlik have prepared a series of peptidyl Michael acceptors with different electron withdrawing groups with different recognition and binding groups as inactivators against papain, a member of the cysteine proteinase family.
However, most of these prior art compounds are reversible inhibitors with diminished efficacy over time and safety problems. The diminished efficacy over time and safety problems are due to these compounds being reversible inhibitors of cysteine proteases that also have a “pro-” form of the cysteine protease; for example, a reversible caspase inhibitor causes more of the pro-caspase in the cell to turn into caspase (due to chemical equilibrium and similar effects) with the end effect being that when the reversible inhibitors release their caspase and leave the cell (or are metabolized), the cell ends up with more caspase than it had to begin with—thus the net effect of the reversible caspase inhibitors over time is the opposite of the desired effect of lowering the caspase levels, causing potentially significant side-effects and safety problems. In addition, some of these inhibitors sometimes bind irreversibly, but when they do, they release a toxic leaving group. Thus, the end result often seen with these types of inhibitors is that, even though they help to ameliorate the disease in the early stages of administration, they ultimately lose most of these effects over time with potentially serious negative side-effects.
The compounds of this invention are directed to a unique new group of compounds with irreversible cysteine protease inhibitory activity, some of which have no leaving groups. Compounds of formula I, II, and IA possess in their structure a Michael acceptor (vinyl groups conjugated to electron withdrawing groups) which confers an irreversible inhibition against cysteine proteases. The molecules can range from 1 to 5 natural or non-natural amino acids and the electrophilic “war head” moieties (e.g, vinyl sulfone, vinyl ester) are attached most notably to aspartic acid although other natural or non-natural amino acids are also possible (see Z, n description).
Wannamaker et al. (WO 01/90063) discloses an ICE inhibitor prodrug for treating IL-1 mediated diseases. However, the prodrug lacks the electrophilic moieties of the compounds of the present invention.
Palmer et al. (U.S. Pat. Nos. 5,976,858 and 6,287,840) described irreversible cysteine protease inhibitors containing vinyl groups conjugated to electron withdrawing groups. Palmer has shown compounds capable to inhibit cathepsins (B, I, S); cruzain and glutathione. However, Palmer showed that his compounds had no activity against cysteine proteases when aspartic acid is used at P1 position (e.g, Asp vinyl sulfone) except in one single case where the activity was below the threshold value for being considered to have any significance by the authors (and was thus discarded for any further development by the authors). This is the opposite of what is observed with the compounds of the present invention.
Similarly, Ng et al. (Org. Biomol. Chem, 2008, 6, 844-847) developed a library of compounds including some vinylsulfone inhibitors to target caspase-3 and -7. However none of the vinylsulfone inhibitors showed potency against caspase-3 and only one molecule showed modest inhibitory activity against caspase-7 according to the authors.
Maria M. M. Santos et al. (European Journal of Med Chem: 46 (2011) 2141 and 45 (2010) 3858) have developed some non-selective aspartic acid vinyl sulfone derivatives with marginal potency. Due to the marginal potency in enzymatic assays, the authors believed that the potency observed was due to an indirect effect on caspase-3 inhibition.
Similarly, Nazif T et al. (EP 1863513, U.S. Pat. No. 7,589,066, PNAS: 2001, 98 (6), 2967-2972) described global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors. Nazif has shown compounds capable to inhibit selectively immunoproteasomes as compared to a constitutive proteasome (10-fold or greater). However, in addition to being selective immunoproteasome inhibitors, Nazif's compounds are apoptosis inducers. The compounds of the present invention are not selective immunoproteasome inhibitors and apoptosis inducers, and in many cases the compounds of the present invention are apoptosis inhibitors.
Given their role in several diseases and conditions, there is a need for compounds capable of selectively targeting either a specific caspase or a group of caspases or a cysteine protease. There is also a need for effective pharmaceutical compositions and methods of treatment for caspase-mediated diseases and cysteine protease-mediated diseases.
The present invention addresses these needs for novel compounds, therapies, new treatment methods, and pharmaceutical compositions.
Additional features of the invention will be apparent from review of the disclosure, tables and description of the invention below.